The invention relates to the modification of plant genomes, particularly to compositions and methods for the excision of nucleotide sequences that have been incorporated into the genome.
Saccharomyces cerevisiae harbor an autonomously replicating 2-xcexc plasmid containing the gene FLPy. FLPy encodes the protein, FLP, a site-specific recombinase acting at defined FLP-recognition targets, FRTs.
Like the cre/lox bacterial system, the yeast FLP/FRT system has been suggested as providing a useful and efficient method for inserting or excising genes in a genome of choice. However, in practice, the yeast FLP nucleic acid sequence (FLPy) does not efficiently express active recombinase in plant cells. For example, in experiments with suspension cells and callus, FLP-transformed maize cells produced only a very low frequency of detectable excision events. See, for example, Lyznik et al. (1993) Nucleic Acids Res. 21:969-975 and the Examples below. Furthermore, it has not previously been demonstrated that the FRT/FLP system functions in monocot plants, or that FRT or FLP are inheritable and functional in the progeny of monocot plants.
It would therefore be very beneficial to provide an FLP nucleic acid sequence that would efficiently produce an effective FLP recombinase in monocot plant cells, particularly in maize cells. Such an effective FLP would permit use of FLP recombinase to selectively and reliably modulate gene excision events in a plant cell genome. It would also be very useful to provide monocot plants stably transformed with FRT nucleic acid sequences, FLP nucleic acid sequences, or both, capable of producing high frequency recombination events, which stably transformed plants pass the FRT/FLP nucleic acid sequences and active recombinase to subsequent generations.
Compositions and methods for modification of plant genomes are provided. The compositions comprise FLP recombinase sequences that have been modified for enhanced expression in plants. The compositions are useful for excision of sequences from the plant genome.
In one embodiment the FLP recombinase is modified for enhanced expression in maize. This specific nucleic acid sequence, moFLP (SEQ ID NO: 1) is based on the native, yeast FLP. moFLP efficiently encodes active FLP recombinase (SEQ ID NO: 2) in monocot plant cells, particularly in maize cells. Monocot plants, particularly maize plants, are stably transformed with moFLP and successfully pass the gene onto succeeding generations with full expression and activity. Maize plants stably carrying FRT sequences are also provided, which FRT-containing plants pass these FRT nucleic acid sequences onto succeeding generations. Transgenic monocot plants expressing moFLP and containing FRT nucleic acid sequences are capable of producing a high frequency of recombination events (i.e., excision).
Hybrid plants are formed by genetically crossing a plant stably expressing moFLP with a plant stably containing FRT sequences. In such hybrid plants, expression of moFLP permits FLP-mediated recombination events at the FRT sequences.
Transformed plants carrying the nucleic acid sequence of at least one FRT site are modified to excise a nucleic acid sequence from between two FRT sites, by inducing expression of moFLP in the plant""s genome. Such expression is induced by transfecting the plant""s cells with moFLP, or by inducing expression of moFLP from the plant""s genome. Induced expression can be via an inducible promoter.